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本研究旨在以当前流行的伪狂犬病病毒变异株(TJ株)为基础,构建携带红色荧光蛋白(RFP)标记细菌人工染色体质粒的重组伪狂犬病病毒(PRV)。通过同源重组和Cre/lox P特异性位点重组的方法构建了重组病毒r PRVTJ-del TK-BAC-RFP株。PCR鉴定和测序分析结果表明,在重组病毒r PRVTJ-del TK-BAC-RFP株的基因组中已插入RFP标记的BAC质粒DNA序列;动力学参数分析表明,重组病毒的生长特性与PRV TJ株相似,尽管复制能力稍有降低,但与亲本株相比并无显著性差异;电镜检测结果显示,重组病毒与亲本病毒PRV TJ株的病毒粒子极其相似,均有囊膜,核心约为80 nm,核衣壳清晰可见。这为进一步研究PRV的变异机制奠定了基础。
The purpose of this study was to construct a recombinant pseudorabies virus (PRV) carrying a red fluorescent protein (RFP) -labeled bacterial artificial chromosome plasmid based on the currently prevailing pseudorabies virus variant (TJ strain). The recombinant virus r PRVTJ-del TK-BAC-RFP strain was constructed by homologous recombination and Cre / lox P site-specific recombination. PCR identification and sequencing analysis showed that RFP-labeled BAC plasmid DNA sequence was inserted into the genome of recombinant PRVTJ-del TK-BAC-RFP strain. Kinetic parameters analysis showed that the growth characteristics of recombinant virus were similar to those of PRV TJ strain Although there was a slight decrease in the replication ability, there was no significant difference compared with the parental strain. The results of electron microscopy showed that the virion of the recombinant virus was very similar to the virion PRV TJ strain, The nucleocapsid is clearly visible. This laid the foundation for further research on the mechanism of PRV mutation.